Our long term objective is to understand the role of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in epithelial cell function. Approximately 70% of patients with Cystic Fibrosis (CF) are homozygous for deltaF508, a mutation that causes a defect in the trafficking of CFTR out of the endoplasmic reticulum. Thus, CF is a "trafficking" disease. However, little is known about the trafficking and polarized sorting of CFTR. Our central hypothesis is that the C-terminal amino acid motif T-R-L plays a key role in sorting or localizing CFTR to the apical membrane of epithelial cells. The T-R-L sequence matches the PDZ interacting domain consensus sequence S/T-X-L/V that binds to proteins containing PDZ domains. This interaction co-localizes proteins within specialized regions of cells including apical and basolateral membranes. In preliminary studies we found that deletion of T-R-L (CFTR-deltaTRL) caused CFTR to become non- polarized, whereas wild type (wt)-CFTR is localized to the apical membrane. Accordingly, our specific aims are to: (1) Test the hypothesis that the C-terminus of CFTR sorts or localizes CFTR to the apical plasma membrane; (2) Elucidate the sorting and trafficking pathways of CFTR; (3) Test the hypothesis that the C-terminus retains CFTR in the apical plasma membrane; and (4) Test the hypothesis that ezrin binding protein 50 (EBP50) and other proteins containing PDZ domains interact with and localized CFTR to the apical membrane. To these ends, several epithelial cell lines, including MDCK and human airway and sweat duct cell lines, will be transfected with green fluorescent protein (GFP)-CFTR expression vectors. Using confocal fluorescent microscopy we will localized GFP- CFTR and, in combination with pulse-chase and selective biotinylation of apical and basolateral membrane proteins, we will elucidate the sorting pathway of CFTR. We will also use a combination of cellular, molecular (i.e., yeast two hybrid interaction trap) and biochemical approaches to determine if CFTR and EBP50 interact, and if this interaction localizes CFTR to the apical membrane. Using the yeast two hybrid interaction trap, we will identify novel proteins that interact with the C-terminus of CFTR. Studies on CFTR trafficking are likely to provide important insights into why epithelial cell function is severely affected in CF and may lead to a treatment for CF. Because a number of other diseases result from defects in protein trafficking (i.e. polycystic kidney disease, Alzheimer's, Tay-Sachs, etc.), we anticipate that our work will be of general interest to investigators in a wide variety of fields.